本文關(guān)鍵詞：HECT類泛素連接酶Smurf1 C426A基因敲入小鼠模型構(gòu)建及表型初步分析　出處：《安徽醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Smurf1 類泛素化修飾 成骨細(xì)胞 表型分析

【摘要】：泛素-蛋白酶體系統(tǒng)調(diào)控著細(xì)胞內(nèi)絕大多數(shù)蛋白的降解。在泛素化過(guò)程中,泛素分子經(jīng)過(guò)泛素活化酶(E1)、泛素結(jié)合酶(E2)和泛素連接酶(E3)的逐級(jí)呈遞,最后結(jié)合到底物蛋白的賴氨酸殘基上,并進(jìn)一步形成泛素鏈,其中E3具有底物識(shí)別的特異性。研究發(fā)現(xiàn)NEDD4超家族HECT類泛素連接酶Smurf1廣泛參與到免疫調(diào)節(jié)、骨動(dòng)態(tài)平衡、腫瘤發(fā)生發(fā)展及神經(jīng)系統(tǒng)發(fā)育等重要的生理、病理過(guò)程。Smurf1可以通過(guò)降解經(jīng)典BMP通路的Smads如Smad1、Smad5、Smad7和BMP受體等負(fù)調(diào)控BMP通路,進(jìn)而上調(diào)了成骨特異基因的轉(zhuǎn)錄。隨后又陸續(xù)鑒定出Smurf1的其它底物分子如Rho A、Runx2、Myd88以及激活形式的MEKK2等。2005年對(duì)Smurf1基因敲除小鼠的研究發(fā)現(xiàn),敲除Smurf1后小鼠骨量會(huì)隨著年齡增長(zhǎng)而增長(zhǎng),骨形成特異增加,Smurf1激活因子CKIP-1的小鼠敲除的研究則發(fā)現(xiàn)其骨密度隨年齡增長(zhǎng)有升高的現(xiàn)象。本課題組前期研究表明,Smurf1能夠催化自身發(fā)生類泛素化Neddylation修飾,該修飾能夠增強(qiáng)Smurf1對(duì)E2的募集作用,提高了其泛素連接酶活性,從而促進(jìn)底物降解。催化反應(yīng)的活性位點(diǎn)位于Smurf1蛋白的N-lobe區(qū)域的426位半胱氨酸。以上結(jié)論均為細(xì)胞和分子水平的研究得出,我們進(jìn)而希望能夠在生理?xiàng)l件下證實(shí)這一結(jié)論,并且探究Smurf1的Neddylation修飾對(duì)生物整體的影響。由于小鼠、發(fā)育過(guò)程、途徑都與人接近,小鼠可作為一種研究模型。我們利用基因工程技術(shù)構(gòu)建了Smurf1 C426A轉(zhuǎn)基因小鼠模型,并對(duì)其表型進(jìn)行了分析研究。首先獲得了小鼠模型后進(jìn)行擴(kuò)繁,并利在小鼠MEF細(xì)胞內(nèi)進(jìn)行體內(nèi)類泛素化修飾實(shí)驗(yàn),確定該模型構(gòu)建成功;分離小鼠頂骨前成骨細(xì)胞進(jìn)行誘導(dǎo)培養(yǎng),探究小鼠成骨細(xì)胞能成骨能力,在動(dòng)物的整體表型上,我們對(duì)小鼠股骨進(jìn)行HE脫鈣切片染色,對(duì)二維切片獲得的骨組織靜態(tài)參數(shù),對(duì)小鼠骨量進(jìn)行了估測(cè),發(fā)現(xiàn)轉(zhuǎn)基因小鼠在骨量上有上升的趨勢(shì)。綜上,本研究在國(guó)際上率先建立了Smurf1 C426A基因敲入的小鼠模型,獲得了關(guān)于Smurf1 C426A基因生理功能的遺傳學(xué)證據(jù),發(fā)現(xiàn)了類泛素化修飾Neddylation與骨形成調(diào)控的聯(lián)系。這些結(jié)果將為HECT類泛素連接酶Smurf1 E3功能研究打下基礎(chǔ),并且加深了對(duì)HECT類泛素連接酶Smurf1 E3功能調(diào)控的認(rèn)識(shí)。
[Abstract]:The ubiquitin proteasome system regulates the degradation of most proteins in the cell. During the process of ubiquification, the ubiquitin molecule passes through the ubiquitin activating enzyme E1). The Ubiquitin binding enzyme E2) and the ubiquitin ligase E3) were presented step by step. Finally, the lysine residues of the protein were bound to the end, and the ubiquitin chain was further formed. E3 has the specificity of substrate recognition. It was found that NEDD4 superfamily HECT ubiquitin ligase Smurf1 is widely involved in immunomodulation and bone homeostasis. Smurf1 can degrade Smads such as Smad1 and Smad5 via classical BMP pathway. Smad7 and BMP receptors negatively regulated the BMP pathway and up-regulated the transcription of osteoblast-specific genes, and then identified other Smurf1 substrates such as Rho Agnon Runx2. Myd88 and activated form of MEKK2 et al. In 2005, a study of Smurf1 gene knockout mice found that the bone mass increased with age after Smurf1 knockout. Bone formation specifically increased the knockout of murf1 activator CKIP-1 in mice and found that bone mineral density increased with age. Smurf1 can catalyze the autogenesis of ubiquitin-like Neddylation modification which can enhance the recruitment of Smurf1 to E2 and the activity of ubiquitin ligase. The active site of catalytic reaction is located at the 426-cysteine in N-lobe region of Smurf1 protein. We then hope to be able to confirm this conclusion under physiological conditions and to explore the effects of Smurf1 Neddylation modification on the overall biology, due to the developmental process in mice. The pathway is similar to that of human, and mice can be used as a research model. We constructed Smurf1 C426A transgenic mice model by genetic engineering. The phenotypes were analyzed and studied. Firstly, the mouse model was obtained, and then expanded and modified in MEF cells. The results showed that the model was successfully constructed. Mouse preparietal osteoblasts were isolated and cultured to explore the osteogenic ability of mouse osteoblasts. In the overall phenotype of animals we stained the femur of mice with HE decalcification sections. The static parameters of bone tissue obtained from two-dimensional sections were estimated, and the bone mass of transgenic mice was found to be increasing. In this study, we first established the mouse model of Smurf1 C426A gene knockin in the world, and obtained the genetic evidence about the physiological function of Smurf1 C426A gene. The relationship between Ubiquitin modified Neddylation and the regulation of bone formation was found. These results will lay a foundation for the study of the function of HECT ubiquitin ligase Smurf1 E3. Furthermore, the function regulation of HECT ubiquitin ligase Smurf1 E 3 was deepened.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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1 舒敬逸;HECT類泛素連接酶Smurf1 C426A基因敲入小鼠模型構(gòu)建及表型初步分析[D];安徽醫(yī)科大學(xué);2016年

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本文編號(hào)：1407657